The human hepatoma line Hep G2 differentiates in vitro, progressing from a phenotype characteristic of fetal liver to one which more closely resembles the adult. This differentiation is denoted by a dramatic lenthening of the doubling time of the cultures, a decline in the synthesis of alphafetoprotein, aldolase A, C and pyruvate kinase K and an increase in the synthesis of albumin, aldolase B and pyruvate kinase L. This proposal describes a detailed analysis of the albumin gene during this differentiation. Messenger RNA levels and changes in transcription will be measured using Northern blots, nuclear run off experiments and quantitative solution hybridization assays. A second phase of this study involves the construction of hybrid genes and transfection into Hep G2 to define the sequences responsible for differentiation dependent alterations. Transient expression assays will be carried out using putative liver specific regulatory sequences attached to CAT, a bacterial gene to be used as a marker. This should allow a reasonably rapid delineation of the sequences necessary for albumin expression. Complementary experiments will monitor transcription at the hybrid gene in stable transfectants with varying copy number. A third phase of this work will use cell free transcription extracts prepared from Hep G2, a subclone, to identify protein transcriptional factors responsible for liver specific albumin gene expression.